Carbohydrate-protein interactions form the basis of a host of biological processes (Clarke A. E. & Wilson I. A., eds. (1988), Carbohydrate-protein interactions (Springer, Heidelberg); and Liener et al (1986) The Lectins: Properties, Functions and Applications in Biology and Medicine (Academia, Orlando, Fla.)). The role of carbohydrates in physiologically relevant recognition has been widely considered (Brandley et al., J Leuk Biol (1986) 40:97; and Sharon et al., Science (1989) 246:227). For example, the cell-surface mediates such processes as cell-cell adhesion (Springer et al, Nature (1990) 346:425) and lymphocyte adhesion through lymphoid tissues (Stoolman, L. M., Cell (1986) 56: 907). Carbohydrate recognition is central to the enzymatic synthesis and degradation of polysaccharides, glycoproteins and glycolipids that play essential roles in metabolism and maintenance of cellular structures.
Tumor associated glycolipids have been reported in fetal tissue and a variety of human cancers, including CML cells (Fukuda et al., J Biol Chem (1986) 261:2376; Magnani et al., J Biol Chem (1982) 257: 14365; Hakomori et al., Biochem Biophys Res Comm (1983) 113:791). This discovery has led to the hypothesis that these structures may be important in many developmental and oncogenic processes (Magnani et al., J Biol Chem (1982) 257:14365). Smaller quantities of most of these carbohydrates can be found in normal human tissue (see Fukushi et al., J Exp Med (1984) 160:506), but until now no function for these structures has been reported.
Adhesion of circulating neutrophils to stimulated vascular endothelium is a primary event of the inflammatory response. Several receptors have been implicated in this interaction, including a family of putative lectins that includes L-selectin (gp90.sup.MEL, Leu8), P-selectin (GMP-140, PADGEM) and E-selectin (ELAM-1) (Gong et al., Nature (1990) 343:757; Johnston et al., Cell (1989) 56:1033; Geoffrey et al., J Cell Biol (1989) 109:2463; Lasky et al., Cell (1989) 56:1045). These receptors each contain a domain with sequence homology to calcium dependent lectins. The receptor gp90.sup.MEL has been demonstrated to recognize a carbohydrate (See Geoffrey et al., J Cell Biol (1989) 109:2463). Endogenous ligands for these receptors are beginning to be characterized (see U.S. Pat. No. 5,143,712 issued Sep. 1, 1992, incorporated herein by reference).
E-selectin or ELAM-1 is particularly interesting because of its transient expression on endothelial cells in response to IL-1 or TNF (Bevilacqua et al., Science (1989) 243:1160). The time course of this induced expression (2-8 h) suggests a role for this receptor in initial neutrophil extravasation in response to infection and injury. Furthermore, Bevilacqua et al (Bevilacqua et al., Proc Natl Acad Sci USA (1987) 84:9238) have demonstrated that human neutrophils or HL-60 cells will adhere to COS cells transfected with a plasmid containing a cDNA encoding for the ELAM-1 receptor.
Several different groups have published papers regarding ELAM-1 ligands which ligands are also referred to as LECAM-2 ligands. Lowe et al (1990) demonstrated a positive correlation between the LECAM-2 dependent adhesion of HL-60 cell variants and transfected cell lines, with their expression of the sialyl Lewis x (sLe.sup.x) oligosaccharide, Neu NAc .alpha.2-3Gal-.beta.1-4(Fuc .alpha.1-3)-GlcNAc. They concluded that one or more members of a family of oligosaccharides consisting of sialylated, fucosylated, lactosaminoglycans are the ligands for the lectin domain of LECAM-2. Phillips et al (1990) used antibodies with reported specificity for sLe.sup.x to inhibit the LECAM-2 dependent adhesion of HL-60 or LEG11 CHO cells to activated endothelial cells. Liposomes containing difucosylated glycolipids with terminal sLe.sup.x structures inhibited adhesion, while those containing nonsialylated Le.sup.x structures were partially inhibitory. Walz et al (1990) were able to inhibit the binding of a LECAM-2-IgG chimera to HL-60 cells with a monoclonal antibody directed against sLe.sup.x or by glycoproteins with the sLe.sup.x structure, but could not demonstrate inhibition with CD65 or CD15 antibodies. Both groups concluded that the sLe.sup.x structure is the ligand for LECAM-2.
P-selectin expressed on activated platelets mediates binding to multiple leukocyte types (Bevilacqua et al, J. Clin. Invest. (1993) 91:379). Endothelial P-selectin also supports leukocyte adhesion.
LECAM-1 is particularly interesting because of its ability to block neutrophil influx (Watson et al., Nature (1991) 349:164-167). It was expressed in chronic lymphocytic leukemia cells which bind to HEV (see Spertini et al., Nature (1991) 349:691-694).
It is believed that HEV structures at sites of chronic inflammation are associated with the symptoms of disease such as rheumatoid arthritis, psoriasis, and multiple sclerosis.
Information regarding the DNA sequences encoding endothelial cell-leukocyte adhesion molecules are disclosed in PCT published application WO90/13300 published Nov. 15, 1990 incorporated herein by reference. The PCT publication cites numerous articles which may be related to endothelial cell-leukocyte adhesion molecules. The PCT publication claims methods of identifying ELAM-ligands, as well as methods of inhibiting adhesion between leukocytes and endothelial cells using such ligands and specifically refers to MILAs which are described as molecules involved in leukocyte adhesion to endothelial cells.
In general, the above publications are directed toward identifying and characterizing endogenous ligands which are carbohydrates.
The development of potent inhibitors of carbohydrate-specific proteins would be of considerable importance in the generation of new therapeutic agents. Oligosaccharides are well positioned to act as recognition molecules due to their cell surface location and structural diversity. However, these carbohydrates are very complex in nature and are expensive and time consuming to synthesize. Chemical synthesis of oligosaccharides requires sophisticated strategies that control product stereochemistry and regiochemistry. Enzymatic synthesis using glycosyltransferases, a viable alternative to chemical synthesis, is limited by the availability of enzymes with appropriate specificities.
Peptides have been suggested as an alternative approach to the synthesis of polysaccharide ligands for carbohydrate-specific receptors (Oldenburg et al, Proc. Natl. Acad. Sci. (1992) 89:5393).
Cell-specific glycopeptide ligands coupled to bioactive materials have been used as a means to deliver said bioactive materials to the selected site (see European Patent Application, Publication No. 63373, published Oct. 27, 1982; and U.S. Pat. No. 4,386,026 issued May 31, 1983 incorporated herein by reference).